U.S. patent application number 11/918581 was filed with the patent office on 2010-01-14 for cancer treatment by combined inhibition of proteasome and telomerase activities.
This patent application is currently assigned to Geron Corporation. Invention is credited to Allison C. Chin, Malcolm A. S. Moore.
Application Number | 20100010064 11/918581 |
Document ID | / |
Family ID | 37115747 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100010064 |
Kind Code |
A1 |
Moore; Malcolm A. S. ; et
al. |
January 14, 2010 |
Cancer treatment by combined inhibition of proteasome and
telomerase activities
Abstract
A method and kit for inhibiting the proliferation of cancer
cells are disclosed, based on a combination of a proteasome
inhibitor and a telomerase inhibitor. When used in cancer therapy,
the two compounds in combination enhance the anti-cancer treatment
efficacy obtained with the proteasome inhibitor alone or the
telomerase inhibitor alone. Preferably, efficacy is supraadditive
or synergistic in nature relative to the combined effects of the
individual agents, with minimal exacerbation of side effects.
Inventors: |
Moore; Malcolm A. S.; (New
York, NY) ; Chin; Allison C.; (Stanford, CA) |
Correspondence
Address: |
King & Spalding LLP
P.O. Box 889
Belmont
CA
94002-0889
US
|
Assignee: |
Geron Corporation
Menlo Park
CA
Memorial Sloan-Kettering Cancer Center
New York
NY
|
Family ID: |
37115747 |
Appl. No.: |
11/918581 |
Filed: |
April 14, 2006 |
PCT Filed: |
April 14, 2006 |
PCT NO: |
PCT/US2006/014137 |
371 Date: |
February 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60671630 |
Apr 15, 2005 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/377; 514/64 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 2310/3145 20130101; C12N 2310/3515 20130101; A61K 31/454
20130101; A61K 45/06 20130101; C12Y 207/07049 20130101; A61K 31/454
20130101; A61K 2300/00 20130101; C12N 2310/11 20130101; C12N
15/1137 20130101 |
Class at
Publication: |
514/44.A ;
435/377; 514/64 |
International
Class: |
A61K 31/711 20060101
A61K031/711; C12N 5/08 20060101 C12N005/08; A61K 31/69 20060101
A61K031/69; A61P 35/00 20060101 A61P035/00; A61K 48/00 20060101
A61K048/00 |
Claims
1. A method for inhibiting the proliferation of cancer cells,
comprising (a) exposing the cells to the proteasome inhibitor
bortezomib, and (b) either proceeding, following, or concomitantly
with step (a), exposing the cells to a telomerase inhibitor.
2. The method of claim 1, wherein the telomerase inhibitor includes
an oligonucleotide having nuclease-resistant intersubunit linkages
and an oligonucleotide sequence effective to bind by
sequence-specific hybridization to a template region of hTR.
3. The method of claim 2, wherein the internucleoside linkages in
the oligonucleotide are selected from N3'.fwdarw.P5'
phosphoramidate and N3'.fwdarw.P5' thiophosphoramidate
linkages.
4. The method of claim 2, wherein the telomerase inhibitor includes
a lipid moiety (i) selected from the group consisting of and fatty
acids, sterols, and derivatives thereof, and (ii) attached
covalently to one end of the oligonucleotide.
5. The method of claim 2, wherein the oligonucleotide is 10-20
bases in length.
6. The method of claim 5, wherein the oligonucleotide is
characterized by: (i) N3'.fwdarw.P5' thiophosphoramidate
internucleoside linkages; (ii) having the sequence identified as
SEQ ID NO: 12; and (iii) a palmitoyl (C16) moiety linked to the 5'
terminus of the oligonucleotide via a glycerol or aminoglycerol
linker.
7. The method of claim 6, wherein the telomerase inhibitor is the
compound designated herein as GRN163L.
8. The method of claim 1, for use in treating a subject having a
cancer, wherein exposing step (a) includes administering the
proteasome inhibitor to the subject in an amount effective, when
the inhibitor is administered alone, to inhibit proliferation of
cancer cells in the subject.
9. The method of claim 8, wherein each exposing step (a) and (b)
includes administering the respective inhibitor to the subject in
an amount effective, when each inhibitor is administered alone, to
inhibit proliferation of cancer cells in the subject.
10. The method of claim 8, wherein the inhibitors are administered
to a subject diagnosed with a cancer selected from the group
consisting of multiple myeloma, non-small-cell lung carcinoma, and
prostate cancer.
11. The method of claim 8, wherein the telomerase inhibitor is the
compound GRN163L, and step (b) includes infusing the telomerase
inhibitor intravenously into the subject, under infusion conditions
effective to produce a blood concentration of the telomerase
inhibitor of between 1 nM and 100 uM.
12. The method of claim 1, wherein the amount of each inhibitor to
which the cells are exposed is effective, by itself, to inhibit
proliferation of the cancer cells.
13. The method of claim 1, wherein said method provides a
supraadditive inhibiting effect relative to the inhibiting effects
of the individual components.
14. A method for enhancing the anti-cancer treatment efficacy of
the proteasome inhibitor bortezomib in a subject, comprising
administering to the subject, before, during, or after
administering the proteasome inhibitor, an oligonucleotide
telomerase inhibitor of the type composed of an oligonucleotide
having nuclease-resistant intersubunit linkages and an
oligonucleotide sequence effective to bind by sequence-specific
hybridization to a template region of hTR.
15. The method of claim 14, wherein the telomerase inhibitor is
administered in an amount effective to inhibit the proliferation of
cancer cells in the subject, when the telomerase inhibitor is
administered alone.
16. The method of claim 14, wherein enhanced treatment efficacy is
evidenced by an increased survival time of the subject, inhibition
of tumor growth in the subject, or a combination thereof.
17. The method of claim 14, wherein the oligonucleotide telomerase
inhibitor is characterized by: (i) N3'.fwdarw.P5'
thiophosphoramidate internucleoside linkages; (ii) having the
sequence identified as SEQ ID NO: 12; and (iii) a palmitoyl (C16)
moiety linked to the 5' terminus of the oligonucleotide via a
glycerol or aminoglycerol linker.
18. The method of claim 17, wherein the oligonucleotide telomerase
inhibitor is GRN163L.
19. The method of claim 18, wherein said administering includes
infusing the oligonucleotide telomerase inhibitor intravenously
into the subject, under infusion conditions effective to produce a
blood concentration of the inhibitor of between 1 nM and 100
.mu.M.
20. The method of claim 14, wherein the two inhibitors are
administered to the subject as a composition containing both
inhibitors.
21. A kit for use in cancer therapy, comprising (a) a dose of the
proteasome inhibitor bortezomib, in an amount of the inhibitor
effective, when administered alone, to inhibit the proliferation of
cancer cells in the subject, and (b) a dose of an oligonucleotide
telomerase inhibitor having nuclease-resistant intersubunit
linkages and an oligonucleotide sequence effective to bind by
sequence-specific hybridization to a template region of hTR.
22. The kit of claim 21, wherein the oligonucleotide is
characterized by: (i) N3'.fwdarw.P5' thiophosphoramidate
internucleoside linkages; (ii) having the sequence identified as
SEQ ID NO: 12; and (iii) a palmitoyl (C16) moiety linked to the 5'
terminus of the oligonucleotide via a glycerol or aminoglycerol
linker.
23. The kit of claim 22, wherein the telomerase inhibitor is the
compound designated herein as GRN163L.
24. The kit of claim 21, wherein the dose of the oligonucleotide
telomerase inhibitor is an amount effective, when administered
alone, to inhibit proliferation of cancer cells in the subject.
25.-26. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention is directed to anticancer treatment, and in
particular to inhibition of tumor growth or cancer-cell
proliferation, by treatment with a telomerase inhibitor in
combination with a proteasome inhibitor.
BACKGROUND
[0002] Although many cancers can be cured by surgical resection,
chemotherapy is often used as an adjunct to surgical therapy, and
it is widely used in the treatment of inoperable or metastatic
malignancy. In view of the continuing high number of deaths each
year resulting from cancer, a continuing need exists to identify
effective and relatively nontoxic therapeutic regimens for use in
anticancer treatment.
[0003] Many effective chemotherapeutic agents have been identified
over the past few decades, and these are generally grouped into
several categories on the basis of their mechanism of action.
Combined-therapy treatments have become more common, in view of the
perceived advantage of attacking the disease via multiple avenues.
In practice, however, many such combinations do not provide even
simple additivity of therapeutic effects.
[0004] Ideally, a combined-drug approach for cancer treatment
should provide a significant boost in efficacy, and/or a
significant reduction in undesired side effects, due to a reduced
dose of the more toxic component, and/or a reduction in the
development of drug-resistance in the cancer being treated.
Particularly desirable are combination therapies which produce
therapeutic results that are supraadditive or synergistic in nature
relative to the effects of the individual agents, with minimal
exacerbation of side effects.
SUMMARY
[0005] The invention includes a method for inhibiting the
proliferation of cancer cells, by (a) exposing the cells to a
proteasome inhibitor of the type effective to inhibit the
chymotrypsin-like activity of proteasomes, and (b) either
proceeding, following, or concomitantly with step (a), exposing the
cells to a telomerase inhibitor. In one embodiment, the amount of
proteasome inhibitor to which the cells are exposed is effective,
by itself, to inhibit proliferation of the cancer cells. In a
further embodiment, the amount of each inhibitor is effective, by
itself, to inhibit proliferation of the cancer cells. Preferably,
the combination provides a enhanced inhibiting effect relative to
either component alone; more preferably, the combination provides a
supraadditive or synergistic effect relative to the combined or
additive effects of the components.
[0006] The telomerase inhibitor may include an oligonucleotide
having nuclease-resistant intersubunit linkages and an
oligonucleotide sequence effective to bind by sequence-specific
hybridization to a template region of hTR. The internucleoside
linkages in the oligonucleotide may be selected from N3'.fwdarw.P5'
phosphoramidate and N3'.fwdarw.P5' thiophosphoramidate linkages.
The telomerase inhibitor may include a lipid moiety, such as a
fatty acid, sterol, or derivative thereof, which is attached
covalently at one end of the oligonucleotide. The oligonucleotide
may be 10-20 bases in length, preferably 13-20 bases in length, and
may have the sequence identified by SEQ ID NO:12
(5'-TAGGGTTAGACAA-3'). One exemplary telomerase inhibitor is the
compound designated herein as GRN163L.
[0007] The proteasome inhibitor may be (a) a synthetic peptide
boronate, such as bortezomib (also known as Velcade.TM. and PS341),
(b) a synthetic peptide aldehyde, such as MG132, MG115, PSI, or
ALLN, (c) a synthetic sulfone tripeptide, (d) a synthetic
epoxoketone, such as epoxomicin, or (e) a microbial
chymotrypsin-like inhibitor, such as lactacystin. One preferred
proteasome inhibitor is bortezomib.
[0008] The method may be used for use in treating a subject having
a cancer, where, preferably, exposing step (a) includes
administering the proteasome inhibitor to the subject in an amount
effective, when administered alone, to inhibit proliferation of
cancer cells in the subject. In a further embodiment, each exposing
step (a) and (b) includes administering the respective inhibitor to
the subject in an amount effective, when administered alone, to
inhibit proliferation of cancer cells in the subject.
[0009] The method may be used in treating a variety of cancers in a
subject. In exemplary embodiments, the method is used in treating
multiple myeloma, non-small-cell lung carcinoma, or prostate cancer
in a subject. Where the telomerase inhibitor is the compound
GRN163L, it may be administered to the subject by intravenous
infusion, under infusion conditions effective to produce a blood
concentration of the inhibitor of between 1 nM and 100 uM.
[0010] In another aspect, the invention is directed to a method for
enhancing the anti-cancer treatment efficacy of the proteasome
inhibitor bortezomib in a subject. The method includes
administering to the subject, before, during, or after
administering bortezomib, an oligonucleotide telomerase inhibitor
of the type composed of an oligonucleotide having
nuclease-resistant intersubunit linkages and an oligonucleotide
sequence effective to bind by sequence-specific hybridization to a
template region of hTR. Preferably, the amount of the telomerase
inhibitor is effective to inhibit the proliferation of cancer cells
in the subject when the telomerase inhibitor is administered
alone.
[0011] The two inhibitors may be administered to the subject as a
composition containing both inhibitors. The enhancement of
treatment efficacy may be evidenced, for example, by an increased
survival time of the subject, or by an inhibition of tumor growth
in the subject, relative to treatment with bortezomib alone. The
method may be used in treating a variety of cancers in a subject.
In exemplary embodiments, the method is used in treating multiple
myeloma, non-small-cell lung carcinoma, or prostate cancer in a
subject.
[0012] The oligonucleotide may be 10-20 bases in length.
Preferably, the oligonucleotide is 13-20 bases in length and
includes the sequence identified by SEQ ID NO: 12
(5'-TAGGGTTAGACAA-3'). An exemplary telomerase inhibitor is the
compound identified as GRN163L, or an analog thereof. This compound
has (i) N3'.fwdarw.P5' thiophosphoramidate internucleoside
linkages; (ii) the sequence identified as SEQ ID NO:12; and (iii) a
palmitoyl (C16) moiety linked to the 5' end of the oligonucleotide
through a glycerol or aminoglycerol linker.
[0013] The invention may also be practiced to identify cancer
patients who are candidates for effective anti-cancer treatment
with a telomerase inhibitor. The candidate patients are those whose
cancer is responding to treatment with bortezomib, or a related
proteasome inhibitor, but for whom combined treatment with a
telomerase inhibitor is desired to enhance the anti-tumor efficacy
of the proteasome inhibitor alone.
[0014] Also disclosed in a kit for use in cancer therapy,
comprising (a) a dose of a proteasome inhibitor of the type capable
of inhibiting the chymotrypsin-like site of proteasomes, in an
amount of the inhibitor effective, when administered alone, to
inhibit the proliferation of cancer cells in the subject, and (b) a
dose of an oligonucleotide telomerase inhibitor having
nuclease-resistant intersubunit linkages, and an oligonucleotide
sequence effective to bind by sequence-specific hybridization to a
template region of hTR. In one embodiment, the telomerase inhibitor
is provided in an amount effective, when administered alone, to
inhibit proliferation of cancer cells in the subject.
[0015] In an exemplary embodiment, the proteasome inhibitor is
bortezomib and the telomerase inhibitor is the compound identified
as GRN163L, or an analog thereof. The latter compound has (i)
N3'.fwdarw.P5' thiophosphoramidate internucleoside linkages in the
oligonucleotide; (ii) the sequence identified as SEQ ID NO: 12; and
(iii) a palmitoyl (C16) moiety linked to the 5' end of the
oligonucleotide through a glycerol or aminoglycerol linker.
[0016] Also provided is a kit comprising a telomerase inhibitor and
bortezomib, for use in therapy, such as the treatment of cancer.
Such therapy preferably comprises administering bortezomib to a
subject, either preceding, following, or concomitantly with
administration of said telomerase inhibitor. The telomerase
inhibitor is preferably a nuclease-resistant oligonucleotide which
binds in a sequence-specific manner to the template region of hTR.
Preferred and/or exemplary inhibitors and cancer indications are as
set out above.
[0017] In a related aspect, the invention provides a kit containing
a dose of an oligonucleotide telomerase inhibitor having
nuclease-resistant intersubunit linkages and an oligonucleotide
sequence effective to bind by sequence-specific hybridization to a
template region of hTR, preferably in an amount effective to
inhibit proliferation of cancer cells in a subject. The kit
preferably includes an insert with instructions for administration;
such an insert may provide a user with one set of instructions for
using the inhibitor in monotherapy and a separate set of
instructions for using the inhibitor in combination with bortezomib
or other proteasome inhibitor. The set of instructions for the
combination therapy may recommend (i) a lower dose of the
telomerase inhibitor, when used in combination with the proteasome
inhibitor, (ii) a lower dose of the proteasome inhibitor, when used
in combination with the proteasome inhibitor, and/or (iii) a
different dosing regimen for one of both inhibitors than would
normally be recommended.
[0018] Also provided is the use of a telomerase inhibitor for
preparation of a medicament for use in treatment of cancer, wherein
said treatment comprises administering said telomerase inhibitor to
a subject in combination with bortezomib. Said treatment may
comprise administering bortezomib to the subject either preceding,
following, or concomitantly with the telomerase inhibitor, which is
preferably a nuclease-resistant oligonucleotide which binds in a
sequence-specific manner to the template region of hTR.
[0019] In a related aspect, the invention provides the use of a
proteasome inhibitor and a telomerase inhibitor, in the manufacture
of a medicament for treating cancer in a subject. Preferred and/or
exemplary inhibitors and cancer indications are as set out
above.
[0020] Further disclosed is the use of a telomerase inhibitor in
the manufacture of a medicament for treating cancer in a subject
who is being treated with a proteasome inhibitor, for purposes of
enhancing the anti-cancer efficacy of a proteasome inhibitor in the
subject. Preferred and/or exemplary inhibitors and cancer
indications are as set out above.
[0021] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1-2 show enhancement of telomerase inhibiting activity
of an NPS oligonucleotide hTR template inhibitor (GRN163) by
conjugation to a lipid (to produce GRN163L), in human myeloma tumor
xenografts (FIG. 1) and liver cells (FIG. 2), respectively, in
mice;
[0023] FIG. 3 illustrates a supraadditive effect on inhibition of
cancer cells achieved by co-administration of the proteasome
inhibitor bortezomib and a telomerase inhibitor, exemplified by
administration of bortezomib and the telomerase inhibitor GRN163L,
in a multiple myeloma xenograft model (see Section IV.A and
Experimental Section D below);
[0024] FIGS. 4A and 4B show measurements of tumor size by
bioluminescence imaging in a disseminated CAG model, with treatment
by bortezomib alone, GRN163L alone, and the two inhibitors together
(4A) and the effect of the individual and combined therapies on
survival time of mice in the disseminated CAG myeloma model (4B)
(see Section IV.A and Experimental Section E below); and
[0025] FIGS. 5A and 5B show measurements of tumor size by
bioluminescence imaging in a disseminated MM1s model, with
treatment by bortezomib alone, GRN163L alone, and the two
inhibitors together (5A) and the effect of the individual and
combined therapies on survival time of mice in the disseminated
MM1s myeloma model (5B) (see Section IV.A and Experimental Section
F below).
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0026] The terms below have the following meanings unless indicated
otherwise.
[0027] A "polynucleotide" or "oligonucleotide" refers to a ribose
and/or deoxyribose nucleoside subunit polymer or oligomer having
between about 2 and about 200 contiguous subunits. The nucleoside
subunits can be joined by a variety of intersubunit linkages,
including, but not limited to, phosphodiester, phosphotriester,
methylphosphonate, P3'.fwdarw.N5' phosphoramidate, N3'.fwdarw.P5'
phosphoramidate, N3'.fwdarw.P5' thiophosphoramidate, and
phosphorothioate linkages. The term also includes such polymers or
oligomers having modifications, known to one skilled in the art, to
the sugar (e.g., 2' substitutions), the base (see the definition of
"nucleoside" below), and the 3' and 5' termini. In embodiments
where the oligonucleotide moiety includes a plurality of
intersubunit linkages, each linkage may be formed using the same
chemistry, or a mixture of linkage chemistries may be used. When an
oligonucleotide is represented by a sequence of letters, such as
"ATGUCCTG," it will be understood that the nucleotides are in
5'.fwdarw.3' order from left to right. Representation of the base
sequence of the oligonucleotide in this manner does not imply the
use of any particular type of internucleoside subunit in the
oligonucleotide.
[0028] The term "nucleoside" includes the natural nucleosides,
including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in
Komberg and Baker, DNA Replication, 2nd Ed. (Freeman, San
Francisco, 1992), and analogs. "Analogs", in reference to
nucleosides, includes synthetic nucleosides having modified
nucleobase moieties (see definition of "nucleobase" below) and/or
modified sugar moieties, e.g., described generally by Scheit,
Nucleotide Analogs (John Wiley, New York, 1980). Such analogs
include synthetic nucleosides designed to enhance binding
properties, e.g., stability, specificity, or the like, such as
disclosed by Uhlmann and Peyman (Chemical Reviews 90:543-584,
1990). An oligonucleotide containing such nucleosides, and which
typically contains synthetic nuclease-resistant internucleoside
linkages, may itself be referred to as an "analog".
[0029] A "nucleobase" includes (i) native DNA and RNA nucleobases
(uracil, thymine, adenine, guanine, and cytosine), (ii) modified
nucleobases or nucleobase analogs (e.g., 5-methylcytosine,
5-bromouracil, or inosine) and (iii) nucleobase analogs. A
nucleobase analog is a compound whose molecular structure mimics
that of a typical DNA or RNA base.
[0030] The term "lipid" is used broadly herein to encompass
substances that are soluble in organic solvents, but sparingly
soluble, if at all, in water. The term lipid includes, but is not
limited to, hydrocarbons, oils, fats (such as fatty acids and
glycerides), sterols, steroids and derivative forms of these
compounds. Preferred lipids are fatty acids and their derivatives,
hydrocarbons and their derivatives, and sterols, such as
cholesterol.
[0031] Fatty acids usually contain even numbers of carbon atoms in
a straight chain (commonly 12-24 carbons) and may be saturated or
unsaturated, and can contain, or be modified to contain, a variety
of substituent groups. For simplicity, the term "fatty acid" also
encompasses fatty acid derivatives, such as fatty or esters.
[0032] The term "hydrocarbon" encompasses compounds that consist
only of hydrogen and carbon, joined by covalent bonds. The term
encompasses open chain (aliphatic) hydrocarbons, including straight
chain and branched hydrocarbons, and saturated as well as mono- and
poly-unsaturated hydrocarbons. The term also encompasses
hydrocarbons containing one or more aromatic rings.
[0033] As used herein, the term "lipid" also includes amphipathic
compounds containing both lipid and hydrophilic moieties.
[0034] The term "substituted" refers to a compound which has been
modified by the exchange of one atom or moiety for another,
typically substitution of hydrogen by a different atom or moiety.
In particular, the term is used in reference to halogenated
hydrocarbons and fatty acids, particularly those in which one or
more hydrogen atoms are substituted with fluorine.
[0035] An "hTR template inhibitor" is a compound that blocks the
template region (the region spanning nucleotides 30-67 of SEQ ID
NO: 1 herein) of the RNA component of human telomerase, thereby
inhibiting the activity of the enzyme. The inhibitor is typically
an oligonucleotide that is able to hybridize to this region.
Preferably, the oligonucleotide includes a sequence effective to
hybridize to a more specific portion of this region, having
sequence 5'-CUAACCCUAAC-3' (SEQ ID NO: 2), spanning nucleotides
46-56 of SEQ ID NO: 1 herein.
[0036] A compound is said to "inhibit the proliferation of cancer
cells" if the proliferation of cells in the presence of the
compound is less than that observed in the absence of the compound.
That is, proliferation of the cells is either slowed or halted in
the presence of the compound. Inhibition of cancer-cell
proliferation may be evidenced, for example, by reduction in the
number of cells or rate of expansion of cells, reduction in tumor
mass or the rate of tumor growth, or increase in survival rate of a
subject being treated.
[0037] Administration of a telomerase inhibitor to a subject is
effective to "enhance the anti-cancer treatment efficacy of a
proteasome inhibitor," if the subject shows a reduced rate of tumor
growth and/or an enhanced survival rate with combined therapy over
therapy with the proteasome inhibitor alone.
[0038] An oligonucleotide having "nuclease-resistant linkages"
refers to one whose backbone has subunit linkages that are
substantially resistant to nuclease cleavage, in non-hybridized or
hybridized form, by common extracellular and intracellular
nucleases in the body; that is, the oligonucleotide shows little or
no nuclease cleavage under normal nuclease conditions in the body
to which the oligonucleotide is exposed. The N3'.fwdarw.P5'
phosphoramidate (NP) or N3'.fwdarw.P5' thiophosphoramidate (NPS)
linkages described below are nuclease resistant.
II. Treatment of Cancer with Proteasome Inhibitors
[0039] Proteasomes control the half-life of many short-lived
regulatory proteins, such as those involved in the cell cycle. The
ubiquitin-proteasome pathway plays an essential role in regulating
the intracellular concentration of specific proteins, thereby
maintaining homeostasis within cells. Inhibition of the 26S
proteasome prevents this targeted proteolysis, which can affect
multiple signaling cascades within the cell. This disruption of
normal homeostatic mechanisms can lead to cell cycle arrest and
cell death. Tumor cells are more susceptible to these effects than
normal cells, in part because they divide more rapidly and in part
because many of their normal regulatory pathways are disrupted.
[0040] Several compounds that inhibit one of more of the
proteolytic activities of proteasomes have been developed for use
in cancer treatment. One general class of proteasome inhibitors is
those that inhibit the chymotrypsin-like activity of proteasomes.
These compounds specifically interact with a key threonine within
the digestive site (catalytic site) of the proteasome, and their
inhibitory activity can be assayed, for example, by the ability of
the compound to block the proteolytic conversion of known
proteasome substrates for chymotrypsin-like activity. Such
substrates are supplied, for example, by BIOMOL International LP,
as can be accessed via the Online Catalog at the BIOMOL website,
under the category "Proteasome Substrates for Chymotrypsin-like
Activity". This inhibitory activity may not be exclusive, and some
compounds known to inhibit the chymotrypsin-like activity of
proteasomes also act to inhibit other proteasome activities, such
as the trypsin-like and peptidyl-glutamyl peptide hydrolyzing
(PGPH) activities of proteasomes.
[0041] Several classes of proteasome inhibitors that inhibit the
chymotrypsin-like activity of proteasomes are known. One important
class includes the peptidyl boronates, such as bortezomib, also
known as Velcade.TM. or PS341, a modified dipeptidyl boronic acid
modified dipeptide boronic acid having the chemical name
[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino)]propy-
l]amino]butyl]boronic acid, and produced by Millenium
Pharmaceuticals.
[0042] Bortezomib is the first proteasome inhibitor to enter
clinical trials for treatment of cancer, and the compound has been
clinically approved and indicated for treatment of multiple
myeloma. In addition, a phase II randomized trial has determined
that bortezomib has significant activity as a single agent with
non-small cell lung cancer (NSCLC) (Fanucchi M P et al., J.
Clinical Oncology 2005; 23:629). Other clinical investigations have
pointed to antitumor effects of bortezomib in breast, colon,
neuroendocrine, and renal cancers, as well as in melanoma, sarcoma,
chronic myelogenoius leukemia, and non-Hodgkin's lymphoma.
Encouraging activity has also been reported in patients with
aggressive forms of lymphoma such as mantle cell lymphoma. Typical
drug dose in cancer treatment is about 1-2 mg/m.sup.2.
[0043] Several other proteasome inhibitors function, at least in
part, by the same mechanism of inhibiting the chymotrypsin-like
activity of proteasomes, and are also suitable for use in the
combination cancer treatment of the invention. These include:
[0044] (a) synthetic peptide aldehydes which reversibly inhibit the
chymotrypsin-like activity, such as MG132, which has been found to
induce apoptosis in human hepatoblastoma HepG2 cells (Emanuele et
al., Int J Oncol., 21:857-865, 2002); MG115, which has been shown
to induce apoptosis in Rat-1 and PC12 cells (Saito et al., Neurosci
Lett, 89:102-107, 1990) and Rock et al., Cell, 78:761-771, 1994);
ritonavir, an inhibitor of HIV-I protease that inhibits the
chymotryptic activity of mouse and human proteasomes (Schmidtke et
al., J Biol Chem, 274(50):35734-35740, 1999), ALLN, which has been
shown to induce apoptosis in insulinoma cells; the proteasome
inhibitors I, II, III and IV (PSI, PSII, PSIII, boronic acid based,
and PSIV), where PSI has been shown to induce apoptosis in human
mesothelioma cells (Sun et al., Cancer Lett, 232(2):161-169, 2006)
and to prevent the expression of plasminogen activator and inhibit
blood vessel formation in embryonic chick chorioallantoic membrane
(Oikawa et al., Biochem Biophys Res Commun, 246:243-248, 1998);
Z-GGL (Wilk and Orlowski, J Neurochem, 40:842-849, 1983); Z-IIF-H;
Z-LLY-COCHO; and peptide glyoxals where an additional ketone moiety
is added at the a position of the peptide aldehydes (Lynas et al.,
Bioorg Med Chem Lett, 8:373-378, 1998),
[0045] (b) synthetic sulfone tripeptides, such as
carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L.sub.3VS) and
related derivatives such as NIP-L.sub.3VS (a nitrophenol
derivative) both of which have been shown to irreversibly inhibit
proteasome activity in vivo (Bogyo et al., PNAS USA, 94:6629-6634,
1997); and NLVS, an irreversible inhibitor of chymotrypsin-like
activity,
[0046] (c) synthetic epoxyketones, such as YU 101
(Ac-hFLFL-epoxide), a potent antiproliferative and
anti-inflammatory agent (Elofsson, et al., Chem Biol,
6(11):811-822, 1999) that is structurally related to epoxomicin,
and
[0047] (d) microbial chymotrypsin-like inhibitors, such as
lactacystin and derivatives, which is a microbial metabolite
isolated from Streptomyces that has been found to inhibit the
formation of vascular endothelial tubes in vitro and prevent the
expression of plasminogen activator and inhibit blood vessel
formation in embryonic chick chorioallantoic membrane (Oikawa et
al., Biochem Biophys Res Commun, 246:243-248, 1998); epoxoketones,
such as epoxomycin, which exhibits in vivo antitumor activity
against B16 melanoma (Hanada et al., J Antibiot, 45(11):1746-1752,
1992) as well as in vivo anti-inflammatory activity (Meng, et al.,
PNAS USA, 96:10403-10408, 1999), eponemycin, which is an antibiotic
that has anti-angiogenic activity (Oikawa et al., Biochem Biophys
Res Commun, 181(3):1070-1076, 1991), and its derivative
dihydroeponemycin; aclarubicin (aclacinomycin), an anthracycline
antibiotic produced by Streptomyces galilaeus having potent
antineoplastic activity, especially in the treatment of leukemias;
tyropeptin A and B, which are isolated from the culture broth of
Kitasatospora sp.; gliotoxin, an immunomodulatory fungal toxin that
has been shown to induce apoptosis in a variety of cells (Ward et
al., J. Biol. Chem., 274:4309, 1999); TMC-86A and B, which were
isolated from Streptomyces sp. fermentation broth (Koguchi et al.,
J Antibiot, 52(12):1069-1076, 1999); TMC-95 isolated from Apiospora
monagnei (Koguchi et al., J Antibiot, 53(2):105-109, 2000); TMC-96,
which was isolated from Saccharothrix sp. fermentation broth
(Koguchi et al., 1999); and PR39, a proline-arginine-rich peptide
antibiotic that was originally isolated from pig intestine (PR11 is
a derivative of PR39 and FALL-39 is the tentative human
counterpart) has been shown to inhibit chymotrypsin-like activity
when studied with fluorogenic peptide substrates and recent studies
have shown that PR39 stimulates angiogenesis and inhibits
inflammatory responses (Li et al., Nature Medicine, 6(1):49-55,
2000) and has been found to inhibit apoptosis in hypoxic
endothelial cells (Wu et al., Circulation, 109(13):1660-1667,
2004).
III. Treatment of Cancer with a Telomerase Inhibitor
[0048] Telomerase is a ribonucleoprotein that catalyzes the
addition of telomeric repeat sequences (having the sequence
5'-TTAGGG-3' in humans) to chromosome ends. See e.g. Blackburn,
1992, Ann. Rev. Biochem. 61:113-129. The enzyme is expressed in
most cancer cells but not in mature somatic cells. Loss of
telomeric DNA may play a role in triggering cellular senescence;
see Harley, 1991, Mutation Research 256:271-282. A variety of
cancer cells have been shown to be telomerase-positive, including
cells from cancer of the skin, connective tissue, adipose, breast,
lung, stomach, pancreas, ovary, cervix, uterus, kidney, bladder,
colon, prostate, central nervous system (CNS), retina and
hematologic tumors (such as myeloma, leukemia and lymphoma).
Targeting of telomerase can be effective in providing treatments
that discriminate between malignant and normal cells to a high
degree, avoiding many of the deleterious side effects that can
accompany chemotherapeutic regimens which target dividing cells
indiscriminately.
[0049] Inhibitors of telomerase identified to date include
oligonucleotides, preferably oligonucleotides having nuclease
resistant linkages, as well as small molecule compounds.
[0050] A. Small Molecule Compounds
[0051] Small molecule inhibitors of telomerase include, for
example, BRACO19
((9-(4-(N,N-dimethylamino)phenylamino)-3,6-bis(3-pyrrolodino
propionamido)acridine (see Mol. Pharmacol. 61(5):1154-62, 2002);
DODC (diethyloxadicarbocyanine), and telomestatin. These compounds
may act as G-quad stabilizers, which promote the formation of an
inactive G-quad configuration in the RNA component of telomerase.
Other small molecule inhibitors of telomerase include BIBR1532
(2-[(E)-3-naphthen-2-yl but-2-enoylamino]benzoic acid) (see Ward
& Autexier, Mol. Pharmacol. 68:779-786, 2005; also J. Biol.
Chem. 277(18):15566-72, 2002); AZT and other nucleoside analogs,
such as ddG and ara-G (see, for example, U.S. Pat. Nos. 5,695,932
and 6,368,789), and certain thiopyridine, benzo[b]thiophene, and
pyrido[b]thiophene derivatives, described by Gaeta et al. in U.S.
Pat. Nos. 5,767,278, 5,770,613, 5,863,936, 5,656,638 and 5,760,062.
One example is
3-chlorobenzo[b]thiophene-2-carboxy-2'-[(2,5-dichlorophenyl
amino)thia]hydrazine, described in U.S. Pat. No. 5,760,062.
[0052] B. Oligonucleotide-Based Telomerase Inhibitors: Sequence and
Composition
[0053] The genes encoding both the protein and RNA components of
human telomerase have been cloned and sequenced (see U.S. Pat. Nos.
6,261,836 and 5,583,016, respectively, both of which are
incorporated herein by reference). Oligonucleotides can be targeted
against the mRNA encoding the telomerase protein component (the
human form of which is known as human telomerase reverse
transcriptase, or hTERT) or the RNA component of the telomerase
holoenzyme (the human form of which is known as human telomerase
RNA, or hTR).
[0054] The nucleotide sequence of the RNA component of human
telomerase (hTR) is shown in the Sequence Listing below (SEQ ID NO:
1), in the 5'.fwdarw.3' direction. The sequence is shown using the
standard abbreviations for ribonucleotides; those of skill in the
art will recognize that the sequence also represents the sequence
of the cDNA, in which the ribonucleotides are replaced by
deoxyribonucleotides, with uridine (U) being replaced by thymidine
(T). The template sequence of the RNA component is located within
the region defined by nucleotides 46-56 (5'-CUAACCCUAAC-3'), which
is complementary to a telomeric sequence composed of about
one-and-two-thirds telomeric repeat units. The template region
functions to specify the sequence of the telomeric repeats that
telomerase adds to the chromosome ends and is essential to the
activity of the telomerase enzyme (see e.g. Chen et al., Cell 100:
503-514, 2000; Kim et al., Proc. Natl. Acad. Sci. USA 98
(14):7982-7987, 2001). The design of antisense, ribozyme or small
interfering RNA (siRNA) agents to inhibit or cause the destruction
of mRNAs is well known (see, for example, Lebedeva, I, et al.
Annual Review of Pharmacology and Toxicology, Vol. 41: 403-419,
April 2001; Macejak, D, et al., Journal of Virology, Vol. 73 (9):
7745-7751, September 1999, and Zeng, Y. et al., PNAS Vol. 100 (17)
p. 9779-9784, Aug. 19, 2003) and such agents may be designed to
target the hTERT mRNA and thereby inhibit production of hTERT
protein in a target cell, such as a cancer cell (see, for example,
U.S. Pat. Nos. 6,444,650 and 6,331,399).
[0055] Oligonucleotides targeting hTR (that is, the RNA component
of the enzyme) act as inhibitors of telomerase enzyme activity by
blocking or otherwise interfering with the interaction of hTR with
the hTERT protein, which interaction is necessary for telomerase
function. See, for example, Villeponteau et al., U.S. Pat. No.
6,548,298.
[0056] A preferred target region of hTR is the template region,
spanning nucleotides 30-67 of SEQ ID NO:1. Oligonucleotides
targeting this region are referred to herein as "hTR template
inhibitors" (see e.g. Herbert et al., Oncogene 21 (4):638-42
(2002).) Preferably, such an oligonucleotide includes a sequence
which is complementary or near-complementary to some portion of the
11-nucleotide region having sequence 5'-CUAACCCUAAC-3', spanning
nucleotides 46-56 of SEQ ID NO: 12.
[0057] Another preferred target region is the region spanning
nucleotides 137-179 of hTR (see Pruzan et al., Nucl. Acids
Research, 30:559-568, 2002). Within this region, the sequence
spanning 141-153 is a preferred target. PCT publication WO 98/28442
describes the use of oligonucleotides of at least 7 nucleotides in
length to inhibit telomerase, where the oligonucleotides are
designed to be complementary to accessible portions of the hTR
sequence outside of the template region, including nucleotides
137-196, 290-319, and 350-380 of hTR. Preferred hTR targeting
sequence are given below, and identified by SEQ ID NOS: 2-22.
[0058] The region of the therapeutic oligonucleotide that is
targeted to the hTR sequence is preferably exactly complementary to
the corresponding hTR sequence. While mismatches may be tolerated
in certain instances, they are expected to decrease the specificity
and activity of the resultant oligonucleotide conjugate. In
particular embodiments, the base sequence of the oligonucleotide is
thus selected to include a sequence of at least 5 nucleotides
exactly complementary to the hTR target, and enhanced telomerase
inhibition may be obtained if increasing lengths of complementary
sequence are employed, such as at least 8, at least 10, at least
12, at least 13 or at least 15 nucleotides exactly complementary to
the hTR target. In other embodiments, the sequence of the
oligonucleotide includes a sequence of from at least 5 to 20, from
at least 8 to 20, from at least 10 to 20 or from at least 10 to 15
nucleotides exactly complementary to the hTR target sequence.
[0059] Optimal telomerase inhibitory activity may be obtained when
the full length of the oligonucleotide is selected to be
complementary to the hTR target sequence. However, it is not
necessary that the full length of the oligonucleotide is exactly
complementary to the target sequence, and the oligonucleotide
sequence may include regions that are not complementary to the
target sequence. Such regions may be added, for example, to confer
other properties on the compound, such as sequences that facilitate
purification. Alternatively, an oligonucleotide may include
multiple repeats of a sequence complementary to an hTR target
sequence.
[0060] If the oligonucleotide is to include regions that are not
complementary to the target sequence, such regions are typically
positioned at one or both of the 5' or 3' termini. Exemplary
sequences targeting human telomerase RNA (hTR) include the
following:
TABLE-US-00001 Region of SEQ ID hTR Targeting Sequence SEQ ID NO: 1
NO: ACATTTTTTGTTTGCTCTAG 160-179 2 GCTCTAGAATGAACGGTGGAAGGCGGCAGG
137-166 3 GTGGAGGCGGCAGG 137-151 4 GGAAGGCGGCAGG 137-149 5
GTGGAAGGCGGCA 139-151 6 GTGGAAGGCGG 141-151 7 CGGTGGAAGGCGG 141-153
8 ACGGTGGAAGGCG 142-154 9 AACGGTGGAAGGCGGC 143-155 10
ATGAACGGTGGAAGGCGG 144-158 11 TAGGGTTAGACAA 42-54 12 CAGTTAGGGTTAG
46-58 13 TAGGGTTAGACA 42-53 14 TAGGGTTAGAC 42-52 15 GTTAGGGTTAG
46-56 16 GTTAGGGTTAGAC 44-56 17 GTTAGGGTTAGACAA 42-56 18 GGGTTAGAC
44-52 19 CAGTTAGGG 50-58 20 CCCTTCTCAGTT 54-65 21 CGCCCTTCTCAG
56-67 22
[0061] The internucleoside linkages in the oligonucleotide may
include any of the available oligonucleotide chemistries, e.g.
phosphodiester, phosphotriester, methylphosphonate, P3'.fwdarw.N5'
phosphoramidate, N3'.fwdarw.P5' phosphoramidate, N3'.fwdarw.P5'
thiophosphoramidate, and phosphorothioate. Typically, but not
necessarily, all of the internucleoside linkages within the
oligonucleotide will be of the same type, although the
oligonucleotide component may be synthesized using a mixture of
different linkages.
[0062] In preferred embodiments, the oligonucleotide has at least
one N3'.fwdarw.P5' phosphoramidate (NP) or N3'.fwdarw.P5'
thiophosphoramidate (NPS) linkage, which linkage may be represented
by the structure: 3'-(-NH--P(.dbd.O)(--XR)--O-)-5', wherein X is O
or S and R is selected from the group consisting of hydrogen,
alkyl, and aryl; and pharmaceutically acceptable salts thereof,
when XR is OH or SH. More preferably, the oligonucleotide includes
all NP or, most preferably, all NPS linkages.
[0063] A particularly preferred sequence for an hTR template
inhibitor oligonucleotide is the sequence complementary to
nucleotides 42-54 of SEQ ID NO: 12 above. The oligonucleotide
having this sequence (TAGGGTTAGACA) and N3'.fwdarw.P5'
thiophosphoramidate (NPS) linkages is designated herein as GRN163.
See, for example, Asai et al., Cancer Research 63:3931-3939 (2003);
Gryaznov et al., Nucleosides Nucleotides Nucleic Acids
22(5-8):577-81 (2003).
[0064] As shown in Table 1 below, this oligonucleotide (first row
of table) inhibits telomerase at low concentrations in a
biochemical assay (FlashPlate.TM.; see Experimental Section). An
alternative 13-mer, having the sequence CAGTTAGGGTTAG,
complementary to nucleotides 46-58 of SEQ ID NO: 1 (fifth row of
table), showed near-equivalent activity in the FlashPlate.TM.
assay. The corresponding NP-linked oligonucleotide, and shorter
(11- and 12-mer) oligonucleotides targeting the same region
(complementary to nucleotides 42-53 and 42-42, respectively, of SEQ
ID NO: 1), showed moderate activity. The effect is clearly
sequence-specific, as shown by the mismatch and non-targeting
sequences in the table.
[0065] The oligonucleotide GRN163 administered alone has shown
inhibitory activity in vitro in cell culture, including epidermoid
carcinoma, breast epithelium, renal carcinoma, renal
adenocarcinoma, pancreatic, brain, colon, prostate, leukemia,
lymphoma, myeloma, epidermal, cervical, ovarian and liver cancer
cells.
[0066] The oligonucleotide GRN163 has also been tested and shown to
be therapeutically effective in a variety of animal tumor models,
including ovarian and lung, both small cell and non-small cell.
TABLE-US-00002 TABLE 1 Inhibition of Telomerase by NPS Oligonucleo-
tides: Biochemical (FlashPlate) Assay Sequence, 5' to 3'
Description IC.sub.50, nM TAGGGTTAGACAA 13-mer (GRN163) 0.045 .+-.
0.007 SEQ ID NO: 12 TAGGTGTAAGCAA Mismatch of GRN163 80 .+-. 31
(SEQ ID NO: 23) sequence TTGTCTAACCCTA Complement of GRN163 1000
.+-. 46 (SEQ ID NO: 24) sequence TAGGGTTAGACAA Duplex of GRN163 8.9
.+-. 3.0 ATCCCAATCTGTT sequence CAGTTAGGGTTAG Alternative targeting
0.049 .+-. 0.007 (SEQ ID NO: 13) 13-mer TAGGGTTAGACA 12-mer;
truncation of 0.36 .+-. 0.2 (SEQ ID NO: 14) GRN163 sequence
TAGGGTTAGAC 11-mer; truncation of 0.85 .+-. 0.35 (SEQ ID NO: 15)
GRN163 sequence GTTAGGGTTAG Alternative targeting 0.51 .+-. 0.13
(SEQ ID NO: 16) 11-mer GTTGAGTGTAG Mismatch of alterna- 177 .+-. 93
(SEQ ID NO: 25) tive targeting 11-mer TAGGGTTAGACAA 13-mer (GRN163
0.7 .+-. 0.1 (SEQ ID NO: 12) sequence) with NP backbone
TAGGTGTAAGCAA Mismatch of GRN163 >1000 (SEQ ID NO: 2) sequence
with NP backbone TTAGGG Telomere repeat unit >1000 (SEQ ID NO:
26) TTTTTTTTTT Oligo-T 10-mer >1000 (SEQ ID NO: 27)
[0067] C. Lipid-Oligonucleotide Conjugates
[0068] Preferably, the oligonucleotide-based enzyme inhibitor
includes at least one covalently linked lipid group (see US Pubn.
No. 2005/0113325, which is incorporated herein by reference). This
modification provides superior cellular uptake properties, such
that an equivalent biological effect may be obtained using smaller
amounts of the conjugated oligonucleotide compared to the
unmodified form. When applied to the human therapeutic setting,
this may translate to reduced toxicity risks, and cost savings.
[0069] The lipid group L is typically an aliphatic hydrocarbon or
fatty acid, including derivatives of hydrocarbons and fatty acids,
with examples being saturated straight chain compounds having 14-20
carbons, such as myristic (tetradecanoic) acid, palmitic
(hexadecanoic) acid, and stearic (octadeacanoic) acid, and their
corresponding aliphatic hydrocarbon forms, tetradecane, hexadecane
and octadecane. Examples of other suitable lipid groups that may be
employed are sterols, such as cholesterol, and substituted fatty
acids and hydrocarbons, particularly polyfluorinated forms of these
groups. The scope of the lipid group L includes derivatives such as
amine, amide, ester and carbamate derivatives. The type of
derivative is often determined by the mode of linkage to the
oligonucleotide, as exemplified below.
[0070] In one exemplary structure, the lipid moiety is palmitoyl
amide (derived from palmitic acid), conjugated through an
aminoglycerol linker to the 5' thiophosphate group of an NPS-linked
oligonucleotide. The NPS oligonucleotide having the sequence shown
for GRN163 and conjugated in this manner (as shown below) is
designated GRN163L herein. In a second exemplary structure, the
lipid, as a palmitoyl amide, is conjugated through the terminal 3'
amino group of an NPS oligonucleotide.
##STR00001##
[0071] As shown in Table 2, conjugation of a single fatty acid-type
lipid significantly increased telomerase inhibitory activity in
cell systems relative to the unconjugated oligonucleotide.
TABLE-US-00003 TABLE 2 Inhibition of Telomerase by Lipid-Conjugated
NPS Oligonucleotides (based on GRN163) Tm (.degree. C.) of duplex
IC.sub.50 in vitro, Lipid Substitution with RNA HT-3 cells, nM none
(GRN163) 70.0 1600 3'-palmitic (GRN163L) 66.5 160 3'-stearic 67.1
140 3'-(bis)stearic ~40 1960 3'-oleic 66.8 930 NH--C.sub.16
(palmitoyl) on 3.sup.rd 62.6 500 5' residue (G) 5'-palmitic 65.5
112 3'-palmitic-5'-palmitic 61.3 ~10000 3'-trityl 66.1 3000
[0072] The effect of lipid conjugation on pharmacokinetics is
illustrated by the data shown in Table 3, below, for a 4 mg/kg dose
administered in rats. Target organ concentrations 6 hours after
administration were also more favorable for GRN163L, with approx.
4-5 .mu.M found in liver, kidney, and fat tissue, 2-3 .mu.M in bone
marrow and spleen, and about 0.5 .mu.M in lymph node. Distribution
of the unlipidated oligonucleotide, GRN163, was primarily to the
kidney (about 18 .mu.M), with only 1 .mu.M or less in the remaining
organ tissues noted above.
[0073] Table 4 presents further data directed to telomerase
inhibition in vitro by GRN163 (unconjugated) and GRN163L
(lipidated) in various cancer cell lines.
TABLE-US-00004 TABLE 3 Comparative Pharmacokinetics of Lipidated
(GRN163L) and Unlipidated (GRN163) NPS Oligonucleotide (Rat, 4
mg/kg dose) GRN163 GRN163L T.sub.1/2.alpha., min 17 20
T.sub.1/2.beta., hrs 65-86 68-72 AUC.sub.0-.infin., .mu.g-hr/g 27
120 C.sub.MAX, .mu.g/ml 16 58 % excreted in 24 h 45 13
TABLE-US-00005 TABLE 4 Comparative Telomerase Inhibitory Activity
of Lipidated (GRN163L) and Unlipidated (GRN163) NPS Oligonucleotide
in vitro GRN163 GRN163L Cell Line IC.sub.50 (.mu.M) IC.sub.50
(.mu.M) HT-3 (Cervical) 1.62 0.3 U251 (Glioblastoma) 1.75 0.17 U87
(Glioblastoma) 0.75 0.11 Hep3B (Hepatoma) 6.5 0.36 HepG2 (Hepatoma)
2.72 0.48 NCI-H522 (Lung) 2.59 0.23 RPMI 8226 2.67 0.38 (Myeloma)
Ovcar5 (Ovarian) 3.74 0.92 DU 145 (Prostate) 1.4 0.15
[0074] The conjugated oligonucleotide GRN163L had significantly
greater telomerase inhibiting activity in vivo than the
unconjugated GRN163, as demonstrated in hepatoma cell xenografts
(FIG. 1) and flank CAG myeloma tumor xenografts (FIG. 2) following
i.v. administration.
[0075] Administration of GRN163L inhibited tumor growth in mice
(A549-luc IV lung metastases model) for at least 4 weeks after i.v.
injection of cancer cells. The dosage was 1 .mu.M biweekly for 5
weeks prior to injection of cancer cells, followed by 5 mg/kg twice
weekly after injection. Controls showed substantial tumor growth,
but none was apparent in the GRN163L-treated mouse.
IV. Combination Therapy with Proteasome and Telomerase
Inhibitors
[0076] Therapeutic benefits for treatment of cancer can be realized
by combining a telomerase inhibitor with other anti-cancer agents.
In one embodiment, the telomerase inhibitor is an hTR template
blocking agent, as described above. Preferably, the combination
provides a enhanced inhibiting effect relative to either component
alone. More preferably, the combination of telomerase inhibition
with chemotherapeutic treatment has a supraadditive effect; that
is, the combined benefit is greater than would be expected simply
from the additive effects of the two therapeutic approaches. This
is demonstrated herein for the combination of a telomerase
inhibitor and the proteasome inhibitor bortezomib
(Velcade.TM.).
[0077] In accordance with the present invention, it has been
discovered that combined exposure of cancer cells to both a
proteasome inhibitor of the type effective to inhibit the
chymotrypsin activity of proteasomes, and a telomerase inhibitor,
enhances the extent to which cell proliferation is inhibited
relative to the proteasome inhibitor alone or the telomerase
inhibitor alone. The effect is seen both for inhibition of cancer
cell growth in vitro, where the inhibition is evidenced by a
reduced rate of cell proliferation, and for in vivo treatment of
cancer in a mammalian subject, where the inhibition is evidenced by
a reduced rate of tumor growth and/or increased survival time of
the subject being treated.
[0078] A. Combined Therapy In Vivo
[0079] In practicing the method of the invention for in vivo
treatment, a subject having a cancer type that is responsive to a
proteasome inhibitor, or a subject currently receiving cancer
therapy with a proteasome inhibitor, is initially identified as a
candidate for the combined therapy. Exemplary cancer indications
include, for example, multiple myeloma, for which the proteasome
inhibitor bortezomib is currently indicated. Recent studies have
shown antitumor activity for this drug in breast, non-small cell
lung (NSCLC), colon, prostate, neuroendocrine, and renal cancers,
as well as in melanoma, sarcoma, chronic myelogenoius leukemia,
non-Hodgkin's lymphoma, and mantle cell lymphoma.
[0080] Thus, an aspect of the invention involves identifying cancer
patients who are candidates for effective anti-cancer treatment
with a telomerase inhibitor. The candidate patients are those whose
cancer is responding to treatment with bortezomib, or a related
proteasome inhibitor, but for whom combined treatment with a
telomerase inhibitor is desired to enhance the anti-tumor efficacy
of the proteasome inhibitor alone.
[0081] The cancer should also be one that is responsive to
cancer-cell inhibition by telomerase inhibition. As noted above,
oligonucleotide telomerase inhibitors, as exemplified by GRN163 and
GRN163L, have shown inhibitory activity in vitro against human
kidney, lung, pancreatic, brain, colon, prostate, breast, leukemia,
lymphoma, myeloma, epidermal, cervical, ovarian and liver cancer
cells, and in vivo, via local and systemic delivery, against human
brain, prostate, lymphoma, myeloma, cervical, lung, and liver
cancer cells. Other preferred targets include small cell lung,
esophageal, head and neck, and stomach cancers.
[0082] In the preferred treatment method, the subject is
administered the proteasome inhibitor, e.g., bortezomib, in an
amount that is effective at inhibiting proliferation of cancer
cells in the subject. The dose administered and the dosing schedule
will follow, for example, known or recommended doses for the
proteasome inhibitor employed, as indicated, for example, in the
drug product insert or published clinical or animal-model data. One
advantage of the present invention is that lower-than-normal doses
of the proteasome inhibitor may be administered, if necessary, due
to the compensating enhancement effect of the telomerase inhibitor.
Such a protocol allows for a reduced dosage of the proteasome
inhibitor, which can have significant toxic effects at higher
dosages.
[0083] Thus, a kit containing a dose of the telomerase inhibitor
could contain a product insert having one set of directions for
using the inhibitor in monotherapy, and another set of directions
for using the inhibitor in a combination therapy with a proteasome
inhibitor, such as bortezomib. The set of instructions for the
combination therapy could recommend (i) a lower dose of the
telomerase inhibitor, when used in combination with the proteasome
inhibitor, (ii) a lower dose of the proteasome inhibitor, when used
in combination with the telomerase inhibitor and/or (iii) a
different dosing regimen for one of both inhibitors than would
normally be recommended.
[0084] The telomerase inhibitor may be administered, before,
during, or after administration of the proteasome inhibitor.
Typically, the two inhibitors are administered in a common dosing
regimen, as described below, and the two inhibitors themselves may
be administered in a combined-drug composition, or separately, for
example, by enteral administration of the proteasome inhibitor and
parenteral administration of the telomerase inhibitor. However, a
dosing regimen in which the telomerase inhibitor is administered
before or after administering the proteasome inhibitor is also
contemplated. For example, a person under treatment with a
proteasome inhibitor may be subsequently placed on a combined
therapy that includes telomerase inhibitor.
[0085] Alternatively, the patient may be initially administered the
proteasome inhibitor, followed one-to-several days later with the
telomerase treatment. In this regimen, the proteasome inhibitor may
function, in part, to sensitize the cancer cells to inhibition by a
telomerase inhibition, e.g., by synchronizing the cell-division
cycle and/or promoting apoptosis in the cells. Preferred dose
levels and dosing schedules are considered further below.
[0086] In one exemplary method, the proteasome inhibitor bortezomib
is administered in combination with a telomerase-inhibitor
oligonucleotide targeted against hTR. FIG. 3, for example, shows
the results of the treatment method in which bortezomib is
administered in combination with the telomerase inhibitor GRN163L,
for the treatment in multiple myeloma in a mouse xenograft model.
Details of this study are given in Experimental Section D below. As
seen from FIG. 3, treatment with the two inhibitors, over a 36-day
treatment period (GRN163 was administered 3 times per week over the
treatment period, and bortezomib, at the times indicated by the
arrows) limited tumor growth to about 4-fold over the pre-treatment
level. With treatment by bortezomib alone, tumor volume increased
nearly 12-fold over the same period (close to that of the untreated
animal group) and with GRN163L alone, tumor growth increased about
8 fold. Thus, the combined effect of the two inhibitors gave an
approximately 300% reduction in tumor size over bortezomib alone,
and about a 200% reduction in tumor size over GRN163L alone.
[0087] A method similar to that just described was carried out with
individual and combined bortezomib and GRN163L therapy in the
disseminated CAG multiple myeloma mouse model, as detailed below in
Experimental Section E, with the results shown in FIGS. 4A and 4B.
In these figures: group 1 represents PBS controls; group 2, GRN163L
administered on a WMF schedule; group 3, GRN163L administered on a
M-F schedule; group 4, IV administered bortezomib; and groups 5 and
6, combination therapy with bortezomib and GRN163L, summarized in
Table 5 below.
[0088] As seen in FIG. 4A, all therapies produced a lower rate of
tumor growth, relative to untreated control. A significant increase
(p<0.01) in survival relative to PBS controls was observed in
each group treated with a single agent (Groups 2-4). GRN163L
administered on either a WMF schedule (Group 2) or M-F schedule
(Group 3) was significantly better than bortezomib (Group 4) alone
(p=0.05 and p<0.001, respectively). Combination chemotherapy
significantly increased the survival of mice compared to the PBS
and single agent controls (FIG. 4B, Groups 5 and 6).
[0089] These experiments also showed that administration of GRN163L
at 36 mg/kg alternating with bortezomib was as effective as
administration of GRN163L at 36 mg/kg tiw (three times a week)
combined with bortezomib (compare Group 5 with Group 6). In other
words, in combination with bortezomib, similar survival benefit
could be achieved with half the dose of GRN163L.
TABLE-US-00006 TABLE 5 Description of groups in disseminated CAG
model M W F M W F Group 1 -- -- -- -- -- -- Group 2 -- G -- G -- G
Group 3 G G G G G G Group 4 Vel -- Vel -- Vel -- Group 5 Vel G Vel
G Vel G +G +G +G Group 6 Vel G Vel G Vel G
[0090] In another method, the combined therapy was applied to
animals in the an animal model having a xenograft of MM1s multiple
myeloma cells, as detailed in Experimental Section F below.
Briefly, GRN163L was administered by intraperitoneal (ip) injection
three time a week at 36 mg/kg for 4 weeks alone or in combination
with 6 doses of bortezomib (0.25 mg/kg). Tumor mass, as measured by
bioluminescence from the tumors, was significantly reduced for both
individual and combined treatment, as seen in FIG. 5A. Similarly,
highest rates of survival were seen with bortezomib alone and with
the combined therapy.
[0091] B. Administration
[0092] The therapeutic protocol for administering such combinations
will depend on various factors including, but not limited to, the
type of cancer, the age and general health of the patient, the
aggressiveness of disease progression, the TRF length (terminal
restriction fragment length; see Section V below) and telomerase
activity of the diseased cells to be treated, and the ability of
the patient to tolerate the agents that comprise the
combination.
[0093] In general, treatment of all carcinoma and hematological
malignancy types is contemplated. In selected embodiments, the
target disease comprises a solid tumor; in other embodiments, the
target disease comprises a hematological malignancy. An exemplary
course of treatment involves multiple doses. Sequence of
combination treatments will be determined by clinical compliance
criteria and/or preclinical or clinical data supporting dose
optimization strategies to augment efficacy or reduce toxicity of
the combination treatment. In general, various combinations of the
telomerase inhibitor and proteasome inhibitor may be employed, used
either sequentially or simultaneously. For multiple dosages, the
two agents may directly alternate, or two or more doses of one
agent may be alternated with a single dose of the other agent, for
example. Simultaneous administration of both agents may also be
alternated or otherwise interspersed with dosages of the individual
agents. The time between dosages may be for a period from about 1-6
hours, to about 6-12 hours, to about 12-24 hours, to about 1-2
days, to about 1-2 wk or longer following the initiation of
treatment. During a course of treatment, the need to complete the
planned dosings may be re-evaluated.
[0094] The compounds may be administered by direct injection of a
tumor or its vasculature. Alternatively, the tumor may be infused
or perfused with the therapeutic compounds using any suitable
delivery vehicle. The compounds may be administered locally to an
affected organ. Systemic administration may also be performed.
Continuous administration may be applied where appropriate; for
example, where a tumor is excised and the tumor bed is treated to
eliminate residual disease. Delivery via syringe or catheterization
is preferred. Such continuous perfusion may take place for a period
from about 1-6 hours, to about 6-12 hours, to about 12-24 hours, to
about 1-2 days, to about 1-2 weeks or longer following the
initiation of treatment. Generally, the dose of the therapeutic
composition via continuous perfusion will be equivalent to that
given by a single or multiple injections, adjusted over a period of
time during which the perfusion occurs.
[0095] The therapeutic agents are administered to a subject, such
as a human patient, in a formulation and in an amount effective to
achieve a clinically desirable result. For the treatment of cancer,
desirable results include reduction in tumor mass (as determined by
palpation or imaging; e.g., by radiography, radionucleotide scan,
CAT scan, or MRI), reduction in the rate of tumor growth, reduction
in the rate of metastasis formation (as determined e.g., by
histochemical analysis of biopsy specimens), reduction in
biochemical markers (including general markers such as ESR, and
tumor specific markers such as serum PSA), and improvement in
quality of life (as determined by clinical assessment, e.g.,
Karnofsky score), increased time to progression, disease-free
survival and overall survival.
[0096] The amount of each agent per dose and the number of doses
required to achieve such effects will vary depending on many
factors including the disease indication, characteristics of the
patient being treated and the mode of administration. Typically,
the formulation and route of administration will provide a local
concentration at the disease site of between 1 nM and 100 .mu.M of
each agent. The physician will be able to vary the amount of the
compounds, the carrier, the dosing frequency, and the like, taking
into consideration such factors as the particular neoplastic
disease state and its severity; the overall condition of the
patient; the patient's age, sex, and weight; the mode of
administration; the suitability of concurrently administering
systemic anti-toxicity agents; monitoring of the patient's vital
organ functions; and other factors typically monitored during
cancer chemotherapy. In general, the compounds are administered at
a concentration that affords effective results without causing
excessive harmful or deleterious side effects.
[0097] In accordance with the invention, the amount of the agent
used in combination with a telomerase inhibitor, especially with
respect to a chemotherapeutic agent such as bortezomib, may be less
than would be required for the agent used in non-combination
therapy.
[0098] C. Formulations
[0099] The pharmaceutical carrier(s) employed may be solid or
liquid. Liquid carriers can be used in the preparation of
solutions, emulsions, suspensions and pressurized compositions. The
compounds are dissolved or suspended in a pharmaceutically
acceptable liquid excipient. Suitable examples of liquid carriers
for parenteral administration include water (which may contain
additives, e.g., cellulose derivatives, preferably sodium
carboxymethyl cellulose solution), phosphate buffered saline
solution (PBS), alcohols (including monohydric alcohols and
polyhydric alcohols, e.g., glycols) and their derivatives, and oils
(e.g., fractionated coconut oil and arachis oil). The liquid
carrier can contain other suitable pharmaceutical additives
including, but not limited to, the following: solubilizers,
suspending agents, emulsifiers, buffers, thickening agents, colors,
viscosity regulators, preservatives, stabilizers and osmolarity
regulators.
[0100] For parenteral administration, the carrier can also be an
oily ester such as ethyl oleate and isopropyl myristate. Sterile
carriers are useful in sterile liquid form compositions for
parenteral administration. Sterile liquid pharmaceutical
compositions, solutions or suspensions can be utilized by, for
example, intraperitoneal injection, subcutaneous injection,
intravenously, or topically. The compositions can also be
administered intravascularly or via a vascular stent.
[0101] The liquid carrier for pressurized compositions can be a
halogenated hydrocarbon or other pharmaceutically acceptable
propellant. Such pressurized compositions may also be lipid
encapsulated for delivery via inhalation. For administration by
intranasal or intrabronchial inhalation or insufflation, the
compositions may be formulated into an aqueous or partially aqueous
solution, which can then be utilized in the form of an aerosol.
[0102] The compositions may be administered topically as a
solution, cream, or lotion, by formulation with pharmaceutically
acceptable vehicles containing the active compound. The
compositions of this invention may be orally administered in any
acceptable dosage including, but not limited to, formulations in
capsules, tablets, powders or granules, and as suspensions or
solutions in water or non-aqueous media. Pharmaceutical
compositions and/or formulations comprising the oligonucleotides of
the present invention may include carriers, lubricants, diluents,
thickeners, flavoring agents, emulsifiers, dispersing aids or
binders. In the case of tablets for oral use, carriers that are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0103] Modes of administration and formulation may be dependent on
the drug and its approved mode of administration. For example, when
the chemotherapeutic agent is bortezomib, i.v. administration is
indicated. When the telomerase inhibitor is GRN163L, formulation in
0.9% sodium chloride (normal saline) and administration by i.v. is
a preferred route, preferably via infusion over 4-8 hours, e.g. a 6
hr infusion. While the lipid-conjugated oligonucleotides described
herein, such as GRN163L, have superior characteristics for cellular
and tissue penetration, these and other compounds may be formulated
to provide further benefit in this area, e.g. in liposome carriers.
The use of liposomes to facilitate cellular uptake is described,
for example, in U.S. Pat. Nos. 4,897,355 and 4,394,448, and
numerous publications describe the formulation and preparation of
liposomes. Liposomal formulations can also be engineered, by
attachment of targeting ligands to the liposomal surface, to target
sites of neovascularization, such as tumor angiogenic regions. The
compounds can also be formulated with additional
penetration/transport enhancers, such as unconjugated forms of the
lipid moieties described above, including fatty acids and their
derivatives. Examples include oleic acid, lauric acid, capric acid,
myristic acid, palmitic acid, stearic acid, linoleic acid,
linolenic acid, dicaprate, tricaprate, recinleate, monoolein
(a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid,
arichidonic acid, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono-
and di-glycerides and physiologically acceptable salts thereof
(i.e., oleate, laurate, caprate, myristate, palmitate, stearate,
linoleate, etc.). Other useful adjuvants include substrates for
transendothelial migration, such as glucose uptake systems for
facilitated egress from the vascular space to the tumor
microenvironment.
V. Measurement of Telomere Length, Telomerase Activity, and/or Cell
Proliferation
[0104] When employing a therapeutic regimen that involves
administration of a telomerase inhibitor, it may be useful to
determine telomere length and/or telomerase activity in a cell or
tissue sample. These parameters can be measured by assays known in
the art. Telomere length can be measured by a flow cytometry method
using fluorescence in situ hybridization, referred to as flow FISH
(see e.g. M. Hultdin et al., Nucleic Acids Res. 26(16):3651-6,
1998; N. Rufer et al., Nature Biotechnology 16:743-7, 1998). Other
methods include terminal restriction fragment (TRF) analysis, in
which genomic DNA is digested with a restriction enzyme having a
four-base recognition sequence not present in telomere repeat
sequences, and the restriction fragments are separated according to
size, e.g. by gel electrophoresis. See, for example, U.S. Pat. No.
5,489,508 (West et al.) and Harley et al., Nature 345:458, 1990.
The West et al. patent also describes methods of measuring telomere
length by a "anchored terminal primer" method and by a modified
Maxam-Gilbert reaction.
[0105] In addition, a more rapid response to a telomerase
inhibiting agent may be predicted for tumor cells having shorter
telomeric DNA, although telomerase has been shown to have other
inhibitory effects independent of telomere length. (e.g. Stewart et
al., PNAS 99:12606, 2002; Zhu et al., PNAS 93:6091, 1996; Rubaiyat
et al., Oncogene 24(8):1320, 2005); and Folini et al., Curr. Pharm.
Design 11 (9):1105, 2005).
[0106] The TRAP assay (see Experimental, below) is a standard
method for measuring telomerase activity in a cell extract system
(Kim et al., Science 266:2011, 1997; Weinrich et al., Nature
Genetics 17:498, 1997). Briefly, this assay measures the amount of
nucleotides incorporated into elongation products (polynucleotides)
formed by nucleotide addition to a labeled telomerase substrate or
primer. The TRAP assay is described in detail in U.S. Pat. Nos.
5,629,154, 5,837,453 and 5,863,726, and its use in testing the
activity of telomerase inhibitory compounds is described in various
publications, including WO 01/18015. In addition, the following
kits are available commercially for research purposes for measuring
telomerase activity: TRAPeze.TM. XK Telomerase Detection Kit
(Intergen Co., Purchase N.Y.); and TeloTAGGG Telomerase PCR ELISA
plus (Roche Diagnostics, Indianapolis Ind.).
[0107] The anticancer activity of the therapeutic combinations can
be evaluated using standard in vitro and in vivo assays. The
ability of a composition to specifically inhibit the growth of
tumor cells can be assayed using tumor cell lines in vitro, or in
xenograft animal models in vivo. A preferred protocol for such
growth curve assays is the short term cell viability assay
described in Asai et al. (2003, cited above). In established
xenograft models of human tumors, the test compound is administered
either directly to the tumor site or systemically, and the growth
of the tumor is followed by physical measurement. A preferred
example of a suitable in vivo tumor xenograft assay is also
described in Asai et al. (2003, cited above). Other examples are
described in Scorski et al., Proc. Natl. Acad. Sci. USA, 94:
3966-3971 (1997) and Damm et al., EMBO J., 20:6958-6968 (2001).
EXPERIMENTAL
A. Preparation and Lipid Conjugation of Oligonucleotide
N3'.fwdarw.P5'Phosphoramidates or N3'.fwdarw.P5'
Thiophosphoramidates
[0108] These compounds may be prepared as described, for example,
in McCurdy et al., Tetrahedron Letters 38:207-210 (1997) or
Pongracz & Gryaznov, Tetrahedron Letters 49:7661-7664 (1999).
The starting 3'-amino nucleoside monomers may be prepared as
described in Nelson et al., J. Org. Chem. 62:7278-7287 (1997) or by
the methods described in Gryaznov et al., US Appn. Pubn. No.
2006/0009636.
[0109] A variety of synthetic approaches can be used to conjugate a
lipid moiety L to the oligonucleotide, depending on the nature of
the linkage selected; see, for example, Mishra et al., Biochim. et
Biophys. Acta 1264:229-237 (1995), Shea et al., Nucleic Acids Res.
18:3777-3783 (1995), or Rump et al., Bioconj. Chem. 9:341-349
(1995). Typically, conjugation is achieved through the use of a
suitable functional groups at an oligonucleotide terminus. For
example, the 3'-amino group present at the 3'-terminus of the NP
and NPS oligonucleotides can be reacted with carboxylic acids, acid
chlorides, anhydrides and active esters, using suitable coupling
catalysts, to form an amide linkage. Thiol groups are also suitable
as functional groups (see Kupihar et al, Bioorg. Med. Chem.
9:1241-1247 (2001)). Various amino- and thiol-functionalized
modifiers of different chain lengths are commercially available for
oligonucleotide synthesis.
[0110] Specific approaches for attaching lipid groups to a terminus
of an NP or NPS oligonucleotide include those described in US Appn.
Pubn. No. 2005/0113325, which is incorporated herein by reference.
In addition to the amide linkages noted above, for example, lipids
may also be attached to the oligonucleotide chain using a
phosphoramidite derivative of the lipid, to produce a
phosphoramidate or thiophosphoramidate linkage connecting the lipid
and the oligonucleotide. The free 3'-amino group of the fully
protected support-bound oligonucleotide may also be reacted with a
suitable lipid aldehyde, followed by reduction with sodium
cyanoborohydride, which produces an amine linkage.
[0111] For attachment of a lipid to the 5' terminus, as also
described in US Appn. Pubn. No. 2005/0113325, the oligonucleotide
can be synthesized using a modified, lipid-containing solid
support. Reaction of 3-amino-1,2-propanediol with a fatty acyl
chloride (RC(O)Cl), followed by dimethoxytritylation of the primary
alcohol and succinylation of the secondary alcohol, provides an
intermediate which is then coupled, via the free succinyl carboxyl
group, to the solid support. An example of a modified support is
shown below, where S--represents a long chain alkyl amine CPG
support, and R represents a lipid.
##STR00002##
[0112] This procedure is followed by synthesis of the
oligonucleotide in the 5' to 3' direction, as described, for
example, in Pongracz & Gryaznov (1999), starting with
deprotection and phosphitylation of the -ODMT group. This is
effective to produce, for example, the following structure, after
cleavage from the solid support:
##STR00003##
[0113] The structure above, when --R is --(CH.sub.2).sub.13CH.sub.3
(palmitoyl), is designated herein as GRN163L.
B. FlashPlate.TM. Assay
[0114] This assay was carried out essentially as described in Asai
et al., Cancer Research, 63:3931 3939 (2003). Briefly, the assay
detects and/or measures telomerase activity by measuring the
addition of TTAGGG telomeric repeats to a biotinylated telomerase
substrate primer. The biotinylated products are captured on
streptavidin-coated microtiter plates, and an oligonucleotide probe
complementary to 3.5 telomere repeats, labeled with 33P, is used
for measuring telomerase products. Unbound probe is removed by
washing, and the amount of probe annealing to the captured
telomerase products is determined by scintillation counting.
C. TRAP Assay
[0115] The ability of a compound to increase or inhibit telomerase
activity in a cell can be determined using the TRAP (Telomeric
Repeat Amplification Protocol) assay, which is described, for
example, in Kim et al., U.S. Pat. No. 5,629,154; Harley et al.,
U.S. Pat. No. 5,891,639; and Harley et al., PCT Pubn. No. WO
2005/000245. Briefly, telomerase-expressing tumor cell lines are
incubated with test compositions, lysed, and treated with a labeled
oligonucleotide telomerase substrate, appropriate primers, and
internal standard for quantitation purposes. Depending on the
telomerase activity of the medium, telomere repeats will be added
to the substrate, to form telomerase extended products. The mixture
is incubated at room temperature, followed by multiple cycles of
PCR. The mixture is separated on a gel, and labeled extension
product is detected and quantitated via comparison with the
internal standard.
D. In Vivo Antitumor Assay Employing GRN163L in Combination with
Bortezomib
[0116] CAG/HSV-TK/GFP/Luc cells (approx. 10.sup.7; TRF length 2.7
Kb) were implanted subcutaneously into NOD/SCID mice one day after
irradiation. Treatment was initiated when tumors reached .about.100
mm.sup.3. Groups of four mice each were treated in accordance with
one of three protocols: (1) GRN163L alone, three times per week
(tiw) at 36 mg/kg for four weeks; (2) bortezomib alone, tiw at
0.125 .mu.g per mouse for three weeks (9 doses); and (3) these
treatments in combination (see FIG. 3). The days when bortezomib
was administered are indicated with arrows in FIG. 3. A fourth
group received saline buffer alone as a control.
[0117] As shown in the Figure, this dose of bortezomib was
minimally effective alone (protocol 2), and this dose of GRN163L
alone (protocol 1) reduced tumor mass by about 30%. In combination
(protocol 3), these agents reduced tumor mass by about 68%. No
significant weight loss was observed.
E. The Effect of Combined Therapy in a Disseminated CAG Model
[0118] The effect of GRN163L in combination with bortezomib was
tested in a disseminated CAG model. The CAG MM cell line (telomere
length 2.7 Kb) was transfected with a retroviral vector encoding a
triple fusion gene for HSV-TK, firefly luciferase and green
fluorescence protein, and whole animal bioluminescence imaging was
undertaken in a disseminated NOD/SCID model with extensive spinal
cord and bone marrow tumor infiltration. NOD/SCID mice were not
irradiated with gamma ray before cell implantation. GRN163L or PBS
was administered by intraperitoneal (i.p.) injections at 36 mg/kg
for 5 weeks (Table 1). Bortezomib was administered intravenously at
a dose of 0.25 mg/kg (Table 1).
[0119] In these experimental settings, bortezomib was used to
target the tumor mass and to help to increase the survival time of
the mice so that GRN163L could be effective on more primitive
cancer cells that would escape the toxic effects of non-specific
chemotherapy in long term. The results are discussed in Section IVA
above, with reference to FIGS. 4A-B.
F. The Effect of Combined Therapy in a Disseminated MM1s Model
[0120] MM1s MM cell line with a short average telomere length (2.5
Kb) was used in a xenograft model to test the effect of GRN163L on
tumor growth in vivo. The MM1s cells were transfected with a
retroviral vector encoding a double fusion gene for firefly
luciferase and green fluorescence protein, and whole animal
bioluminescence imaging was undertaken in a disseminated NOD/SCID
model with extensive spinal cord and bone marrow tumor
infiltration. NOD/SCID mice were irradiated with 300 rads of gamma
ray before cell implantation. GRN163L was administered by
intraperitoneal (i.p.) injections three times in two weeks at 36
mg/kg for 4 weeks alone or in combination with 6 doses of
bortezomib (0.25 mg/kg). The results are discussed in Section IVA
above, with reference to FIGS. 5A-B.
[0121] Although the invention has been described with respect to
particular embodiments and applications, those skilled in the art
will appreciate the range of applications and methods of the
invention disclosed herein.
Sequence CWU 1
1
271554RNAHomo sapiens 1ggguugcgga gggugggccu gggaggggug guggccauuu
uuugucuaac ccuaacugag 60aagggcguag gcgccgugcu uuugcucccc gcgcgcuguu
uuucucgcug acuuucagcg 120ggcggaaaag ccucggccug ccgccuucca
ccguucauuc uagagcaaac aaaaaauguc 180agcugcuggc ccguucgccu
cccggggacc ugcggcgggu cgccugccca gcccccgaac 240cccgccugga
gccgcggucg gcccggggcu ucuccggagg cacccacugc caccgcgaag
300aguugggcuc ugucagccgc gggucucucg ggggcgaggg cgagguucac
cguuucaggc 360cgcaggaaga ggaacggagc gagucccgcc gcggcgcgau
ucccugagcu gugggacgug 420cacccaggac ucggcucaca caugcaguuc
gcuuuccugu uggugggggg aacgccgauc 480gugcgcaucc gucaccccuc
gccggcagug ggggcuugug aacccccaaa ccugacugac 540ugggccagug ugcu
554220DNAArtificial SequenceSynthetic Oligonucleotide 2acattttttg
tttgctctag 20330DNAArtificial SequenceSynthetic Oligonucleotide
3gctctagaat gaacggtgga aggcggcagg 30414DNAArtificial
SequenceSynthetic Oligonucleotide 4gtggaggcgg cagg
14513DNAArtificial SequenceSynthetic Oligonucleotide 5ggaaggcggc
agg 13613DNAArtificial SequenceSynthetic Oligonucleotide
6gtggaaggcg gca 13711DNAArtificial SequenceSynthetic
Oligonucleotide 7gtggaaggcg g 11813DNAArtificial SequenceSynthetic
Oligonucleotide 8cggtggaagg cgg 13913DNAArtificial
SequenceSynthetic Oligonucleotide 9acggtggaag gcg
131016DNAArtificial SequenceSynthetic Oligonucleotide 10aacggtggaa
ggcggc 161118DNAArtificial SequenceSynthetic Oligonucleotide
11atgaacggtg gaaggcgg 181213DNAArtificial SequenceSynthetic
Oligonucleotide 12tagggttaga caa 131313DNAArtificial
SequenceSynthetic Oligonucleotide 13cagttagggt tag
131412DNAArtificial SequenceSynthetic Oligonucleotide 14tagggttaga
ca 121511DNAArtificial SequenceSynthetic Oligonucleotide
15tagggttaga c 111611DNAArtificial SequenceSynthetic
Oligonucleotide 16gttagggtta g 111713DNAArtificial
SequenceSynthetic Oligonucleotide 17gttagggtta gac
131815DNAArtificial SequenceSynthetic Oligonucleotide 18gttagggtta
gacaa 15199DNAArtificial SequenceSynthetic Oligonucleotide
19gggttagac 9209DNAArtificial SequenceSynthetic Oligonucleotide
20cagttaggg 92112DNAArtificial SequenceSynthetic Oligonucleotide
21cccttctcag tt 122212DNAArtificial SequenceSynthetic
Oligonucleotide 22cgcccttctc ag 122313DNAArtificial
SequenceSynthetic Oligonucleotide 23taggtgtaag caa
132413DNAArtificial SequenceSynthetic Oligonucleotide 24ttgtctaacc
cta 132511DNAArtificial SequenceSynthetic Oligonucleotide
25gttgagtgta g 11266DNAArtificial SequenceSynthetic Oligonucleotide
26ttaggg 62710DNAArtificial SequenceSynthetic Oligonucleotide
27tttttttttt 10
* * * * *